Abstract
ABSTRACTThe emergence of Klebsiella pneumoniae carbapenemases (KPCs), β-lactamases that inactivate “last-line” antibiotics such as imipenem, represents a major challenge to contemporary antibiotic therapies. The combination of ceftazidime (CAZ) and avibactam (AVI), a potent β-lactamase inhibitor, represents an attempt to overcome this formidable threat and to restore the efficacy of the antibiotic against Gram-negative bacteria bearing KPCs. CAZ-AVI-resistant clinical strains expressing KPC variants with substitutions in the Ω-loop are emerging. We engineered 19 KPC-2 variants bearing targeted mutations at amino acid residue Ambler position 179 in Escherichia coli and identified a unique antibiotic resistance phenotype. We focus particularly on the CAZ-AVI resistance of the clinically relevant Asp179Asn variant. Although this variant demonstrated less hydrolytic activity, we demonstrated that there was a prolonged period during which an acyl-enzyme intermediate was present. Using mass spectrometry and transient kinetic analysis, we demonstrated that Asp179Asn “traps” β-lactams, preferentially binding β-lactams longer than AVI owing to a decreased rate of deacylation. Molecular dynamics simulations predict that (i) the Asp179Asn variant confers more flexibility to the Ω-loop and expands the active site significantly; (ii) the catalytic nucleophile, S70, is shifted more than 1.5 Å and rotated more than 90°, altering the hydrogen bond networks; and (iii) E166 is displaced by 2 Å when complexed with ceftazidime. These analyses explain the increased hydrolytic profile of KPC-2 and suggest that the Asp179Asn substitution results in an alternative complex mechanism leading to CAZ-AVI resistance. The future design of novel β-lactams and β-lactamase inhibitors must consider the mechanistic basis of resistance of this and other threatening carbapenemases.
Highlights
All 19 amino acid variants at Ambler position 179 in Klebsiella pneumoniae carbapenemases (KPCs)-2 -lactamase were engineered. To determine whether these single substitutions alter protein expression, immunoblots using whole-cell preparations and periplasmic extracts were probed with an anti-KPC-2 polyclonal antibody (Ab) that is sensitive and specific and maps to three main linear epitopes of KPC-2 [9]
Desulfation of avibactam is known to occur with KPC-2 [18, 19], and we show here that it does not occur in the Asp179Asn variant (Fig. 5A and S1)
-lactamase that is notable is the KPC-2 Asp179Asn variant, as E. coli expressing blaKPC-2 Asp179Asn demonstrated resistance to ceftazidime and to other -lactams and -lactam–-lactamase inhibitor combinations, including ceftazidime-avibactam. This laboratory analysis closely recapitulates the clinical observations being reported showing the emergence of KPC variants resistant to ceftazidime-avibactam [15]
Summary
University California Medical Center, Los Angeles, California, USAl; Division of Infectious Diseases, Keck School of Medicine at USC, Los Angeles, California, USAm. The emergence of Klebsiella pneumoniae carbapenemases (KPCs), -lactamases that inactivate “last-line” antibiotics such as imipenem, represents a major challenge to contemporary antibiotic therapies. We focus on the CAZ-AVI resistance of the clinically relevant Asp179Asn variant. This variant demonstrated less hydrolytic activity, we demonstrated that there was a prolonged period during which an acyl-enzyme intermediate was present. Using mass spectrometry and transient kinetic analysis, we demonstrated that Asp179Asn “traps” -lactams, preferentially binding -lactams longer than AVI owing to a decreased rate of deacylation
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